Polypeptides are increasingly being used as medicaments for the treatment of diseases within all major therapy areas. Treatment of diabetes by chronic insulin administration has been practised for more than 80 years, and therapeutic applications of polypeptides within growth disorders and cancer also have been practised for many years.
Economical processes for the large scale production of polypeptides with a purity sufficiently high for therapeutic applications are crucial for further polypeptide-based therapies to reach the mass market and for the existing therapies to become more widely used.
Purification of a polypeptide from a mixture is a step which is normally used several times during the overall manufacturing process for a therapeutic polypeptide. Reverse phase high pressure liquid chromatography (RP-HPLC) is the preferred method for industrial high resolution separation of polypeptides, and the method has proven versatile for the large scale purification of many polypeptides.
Since polypeptides for therapeutic use are to be highly purified in order not to cause adverse events upon administration to the patient, it is quite common to use several chromatographic purification steps in the manufacturing process. The stationary phase of chromatographic columns in manufacturing plants are expensive and they are thus used for several chromatographic cycles. However, over time the performance of the chromatographic stationary phase declines, i.e. pressure drop over the column increases prohibitively and the separation factor is impaired. This has been attributed to the gradual build-up of deposits.
The problem has been suggested to be overcome by a regeneration process comprising alkaline buffers (J. Chrom. 461, 1989, 45-61), e.g. pH 7.4 and high concentration of organic modifier.
Brange et al. (J. Pharm. Sci. 86 (1997) 517-525) discloses dissolving insulin fibrils in acid and in base.
For many years the problem has been alleviated by regenerating the chromatographic stationary phase with alkaline solution, e.g. 0.1 molar sodium hydroxide (vide Liliedahl, “Twelve years of silica-based HPLC purification with focus on peptides”, at Tides 2000, 10 May 2000 in Las Vegas, USA). This regeneration process may increase the lifetime of silica used for purifying insulin to between 100 to 600 cycles. However, silica materials are not stable when exposed to harsh alkaline conditions, and especially substituted silica materials may not be amenable to regeneration by alkaline solutions. Economically viable processes for purifying pharmaceuticals such as therapeutic polypeptides must include a regeneration process which does not degrade the chromatographic stationary phase.
A general and complex process for regenerating particulate materials (clay, sand, silica etc.) from a wide variety of sources has been disclosed in WO 00/61493. It is a 5 step process comprising contacting the material with a) an extractant of organic material, b) an oxidizing agent, followed by c) an acid solution, d) heating the material and e) recovering the material. The process is cumbersome and not amenable for implementation in a chromatographic purification plant.
There is a need in the art for more efficient ways of regenerating chromatographic stationary phases so as to increase the lifetime of these expensive raw materials and prevent the pressure drop over chromatographic columns to rise. Especially, regeneration processes which are suited for in-situ regeneration of chromatographic stationary phases in manufacturing plants are needed.